Project Summary It has been estimated that 5.6% of adult Americans have Barrett?s esophagus (BE), a major risk factor for esophageal adenocarcinoma. To prevent this cancer, patients with BE are advised to have regular endoscopic surveillance for dysplasia, and to have that dysplasia treated with radiofrequency ablation (RFA). Unfortunately, surveillance has not prevented deaths from esophageal cancer, and Barrett?s metaplasia recurs frequently after RFA. Surveillance failures and metaplasia recurrences might be due to subsquamous intestinal metaplasia (SSIM), a condition in which metaplastic glands are located in the lamina propria under a layer of squamous epithelium that hides them from the endoscopist and shields them from destruction by RFA. SSIM initially was considered a side effect of endoscopic ablation, but recent studies show that SSIM is present in the large majority of Barrett?s patients who have not had ablation procedures. This highly prevalent SSIM might be the source of tumors missed by endoscopic surveillance, and the nidus for recurrent metaplasia after RFA. Thus, SSIM appears to be a frequent and important condition that limits the efficacy of endoscopic surveillance for the millions of patients with BE, and that thwarts endoscopic attempts to eradicate BE and prevent its progression to cancer. Epithelial-mesenchymal transition (EMT) is the process in which epithelial cells acquire mesenchymal characteristics including cell migration. In BE, EMT could enable metaplastic Barrett?s epithelial cells to migrate into the lamina propria underneath adjacent squamous epithelium, resulting in SSIM. In our rat model in which we induce reflux esophagitis by creating an esophago-jejunostomy, our preliminary data strongly suggest that jejunal epithelial cells adjacent to ulcerated esophageal squamous mucosa undergo EMT, which contributes to the development of a columnar-lined esophagus with features of Barrett?s metaplasia including SSIM. Gastroesophageal reflux causes esophageal inflammation and production of reactive oxygen species, conditions that can activate hypoxia inducible factors (HIFs). Our preliminary data show that Barrett?s epithelial cells exposed to acid and bile salts exhibit a strong and sustained increase in nuclear HIF-1? and HIF-2?. In patients with BE, we also show that HIF-1? and HIF-2? levels in their Barrett?s metaplasia rise when the esophagus is perfused with acid or bile salts. HIFs can promote EMT by causing cells to secrete vascular endothelial growth factor (VEGF), which binds the cells? VEGF receptors in an autocrine fashion to induce VEGF signaling that triggers EMT. Our preliminary data show that acid and bile salts induce autocrine VEGF signaling and EMT features in Barrett?s cell lines. Therefore, we hypothesize that reflux-induced activation of HIFs in Barrett?s epithelial cells causes VEGF secretion with autocrine VEGF signaling, which initiates the EMT program and causes SSIM. The aims of this study are to elucidate the mechanism(s) whereby acid and bile salts activate HIFs to cause VEGF production, and to explore the role of autocrine VEGF signaling in the EMT program induced by acid and bile salts in Barrett?s cells in vitro, and in BE patients with reflux esophagitis.